activity of a purified his-tagged 3c-like proteinase from the coronavirus infectious bronchitis virus：活性的纯化his标签的3c像从冠状病毒传染性支气管炎病毒蛋白酶.pdf

Virus Research 60 (1999) 137–145 Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus K.W. Tibbles 1,*, D. Cavanagh 2, T.D.K. Brown 1 1 Diision of Virology, Department of Pathology, Uniersity of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK 2 Institute for Animal Health, Compton Laboratory, Newbury, Berks RG20 7NN, UK Received 23 October 1998; accepted 12 January 1999 Abstract Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the proc